Yeast eIF1 and eIF5 interact with eIF4G in vitro.
We previously found that both yeast eIF4G isoforms eIF4G1 and eIF4G2 could bind eIF5 in vitro and localized the eIF5 binding domain of eIF4G2 to its C-terminal half, which is homologous to the middle domain of mammalian eIF4G containing HEAT motifs (6
). To examine if yeast eIF4G binds eIF1, we first conducted in vitro protein binding assays. Purified yeast eIF1 or bovine serum albumin (BSA) as a control was immobilized on a microtiter plate and incubated with recombinant yeast eIF4G1 purified from insect cells. Binding of eIF4G1 to the immobilized proteins was detected via anti-eIF4G1 antibodies and alkaline phosphatase-coupled secondary antibodies as described in Materials and Methods. As shown in Fig. , eIF4G1 specifically bound to the immobilized eIF1, but not BSA, on the microtiter plate. To examine which domain of eIF4G binds eIF1, N- and C-terminal halves of eIF4G2 (eIF4G21-513
) containing the eIF4E/PABP- or eIF4A-binding domain, respectively, were purified and adsorbed to the plate. Binding of GST-fused derivatives of eIF1 or eIF5 was then detected via anti-GST antibodies. As shown in Fig. , GST-eIF1 bound specifically to eIF4G2439-914
(column 6), but not to eIF4G21-513
(column 5). Similar results were obtained for GST-eIF5 (columns 8 and 9), as observed previously (6
We also tested whether the binary complexes observed in Fig. were stable enough to be isolated by affinity purification. eIF4G2439-914 was mixed with purified GST alone, GST-eIF1, or GST-eIF5, and the resulting complexes were repurified on a glutathione-Sepharose column and visualized by silver staining. As shown in Fig. , bottom panel, the eIF4G2 segment specifically copurified with GST-eIF1 (lane 2) and GST-eIF5 (lane 4) but not with GST alone (lane 1). Treatment of the binary complexes with RNase A did not abolish these interactions (lanes 3 and 5), excluding the possibility that they are mediated by RNA bound fortuitously to a component of the binding reaction. We conclude that the C-terminal half of eIF4G, encompassing the HEAT domain, can bind directly to both eIF1 and eIF5.
The ratios of eluted GST fusion protein to coeluted eIF4G439-915
suggest that the interactions involved in tethering the eIF4G fragment to GST-eIF1 or GST-eIF5 are relatively weak. Since all proteins for the experiment presented in Fig. were used at initial concentrations of 1.25 μM, the results suggest an apparent equilibrium dissociation constant (Kd
) of ~10 μM. In further experiments, we also used different amounts of GST-eIF1 and GST-eIF5241-405
(the minimal segment for eIF4G binding, known as B6 [6
]) tethered to the glutathione resin for the binding assays with 35
) (Fig. , top panel). Plotting the fraction of bound 35
against the concentration of GST fusion protein was consistent with a hyperbolic curve (Fig. ), and apparent Kd
values were deduced to be 9.4 ± 2.3 μM for GST-eIF1 and 8.2 ± 1.8 μM for GST-eIF5241-405
. It should be noted, however, that these calculations neglect the loss of the eIF4G2 fragment that occurs during the washing steps. These values therefore represent upper limits for the Kd
value of the respective interactions.
The entire HEAT domain and flanking residues of yeast eIF4G2 are required for optimal binding of eIF1 and eIF5.
The top panel of Fig. shows the location of the 10 α-helices that constitute the HEAT domain of yeast eIF4G2 between residues 557 and 812. Two consecutive α-helices (e.g., 1a and 1b) correspond to a single HEAT repeat and form one pair of antiparallel helices as a part of the entire structure (2
). The eIF4G2439-914
segment used in the previous experiments contains additional segments flanking the deduced HEAT domain (Fig. , row 2). To narrow down the binding domains for eIF1 and eIF5, we tested GST-eIF1 and GST-eIF5241-405
for binding to differently truncated eIF4G2 segments synthesized and labeled with [35
S]methionine in a rabbit reticulocyte lysate. Figure shows the results of these experiments.
FIG. 2. Effect of deletions (A) and point mutations (B) on eIF4G2 binding to eIF1 and eIF5. The binding of GST (column C, lane 2), GST-eIF1 (column 1, lane 3), or GST-eIF5241-405 (column 5, lane 4) (20 μg each) to different derivatives of 35S-eIF4G2 synthesized (more ...)
First, we found that 35S-eIF4G2439-914 (row 2), but not 35S-eIF4G21-513 (row 1), efficiently bound to both GST-eIF1 and GST-eIF5241-405, confirming that the C-terminal half of eIF4G is responsible for binding to eIF1 or eIF5. Further N-terminal truncation to residue 514 or beyond abolished the binding to both proteins (rows 3 and 4). This result placed the N-terminal boundary of the binding domains for eIF1 and eIF5 between residues 440 and 513, N terminal to all of the HEAT repeats (see Discussion). The C-terminal deletions in eIF4G2439-914 extending up to residue 577, between the first and second HEAT repeats (rows 5 to 7), reduced binding of the eIF4G2 peptides to GST-eIF5241-405 by 40% or less. However, the same C-terminal deletions (rows 5 to 7) reduced the binding of eIF4G2 to GST-eIF1 by 70 to 88% of that given by eIF4G2439-914 (row 2). Thus, the segment C terminal to the HEAT repeats is required for optimal binding of both eIF5 and eIF1 but is particularly important for eIF1 binding. It is interesting that the smallest eIF4G2439-577 segment still showed a weakened but specific binding to GST-eIF1 and GST-eIF5241-405 (row 7), suggesting that the primary eIF1/eIF5-binding site may be located within the N-terminal third of eIF4G2439-914.
, and tif4632-8
mutations alter four, two, and four amino acids, respectively, in the C-terminal half of eIF4G2 (Fig. ) and have been shown to reduce its binding to eIF4A in vitro and in vivo (26
). According to the structure solved for the mammalian eIF4GII HEAT domain, most, if not all, of these mutations map in the hydrophobic core and almost certainly destabilize protein structure (21
). Thus, we used these mutations to examine the effect of disrupting the entire HEAT domain structure. We found that all these mutations reduced the binding of eIF4G2439-914
to eIF1 and eIF5 (rows 8 to 10). In particular, tif4632-1
, altering the entire HEAT domain, reduced the interaction with eIF5 by 81% (row 8) and tif4632-8
, altering the N-terminal extension and a part of the HEAT domain, reduced both the interactions with eIF1 and eIF5 by 75 to 78% (row 10). Based on all these results, we conclude that the entire HEAT repeat domain and flanking residues between 439 and 914 are required for optimal binding of both eIF1 and eIF5.
The interactions of eIF4G with eIF1 and eIF5 are mutually exclusive, whereas eIF1 can bind simultaneously to eIF4G and eIF3c.
We reported previously that eIF1 and eIF5 can interact simultaneously with eIF3c (3
). Having observed that eIF1 and eIF5 interact with the eIF4G domain at binding sites close to each other (Fig. ), we wished to examine whether eIF1 and eIF5 can bind simultaneously to eIF4G. We also asked whether eIF1 could bind simultaneously to eIF4G and eIF3c, as shown previously for the eIF5 CTD (6
To test if eIF1 and eIF5 can bind to eIF4G2 simultaneously, we first examined if the purified eIF4G2439-914 can serve as a bridge between eIF1 and eIF5 (Fig. ). We used a purified eIF3c segment (eIF3c1-156), known to form a bridge between eIF1 and eIF5, as a positive control in the present experiments. As shown in Fig. , lanes 5 and 6, we found that eIF4G2439-914 did not enhance the relatively weak interaction between GST-eIF5241-405 and 35S-eIF1 (bottom panel), even though the purified eIF4G segment can bind efficiently to GST-eIF5241-405 (top panel). By contrast, purified eIF3c1-156 enhanced the GST-eIF5241-405/eIF1 interaction as reported previously (Fig. , lanes 9 and 10). The weak interaction between eIF5 and eIF1 was not prevented by the binding of eIF4G to eIF5 (lanes 5 and 6), suggesting that the eIF5 CTD can bind concurrently to eIF1 and eIF4G.
FIG. 3. Relationship between binary interactions between eIF1, eIF4G, eIF3c, and eIF5-CTD. (A) Protein linkage between factors involved in 43S and 48S complex formation. Circles indicate eIFs, with numbers identifying each. Shaded circles indicate eIFs involved (more ...)
We then examined a reverse combination regarding which factor is fused to GST and present in excess. As shown in lane 5 of Fig. , the N-terminal fusion of eIF1 to a GST-fused protein abolished its ability to bind eIF5 (manuscript in preparation for our detailed analyses on the eIF1/eIF5 interaction [C. R. Singh, H. He, M. Ii, and K. Asano, unpublished data]). We found that the eIF3c segment, but not the eIF4G2 segment, bridged the interaction between GST-eIF1 and 35S-eIF5241-405 (Fig. , lanes 6 and 10), indicating again that the eIF4G segment does not serve as a bridge between eIF1 and eIF5.
The amount of the eIF4G2 segment (~2 μg) tethered to GST-eIF5241-405 or -eIF1 (lanes 6 in top panels of Fig. and C) should be sufficient to bind 35S-eIF1 or -eIF5241-405, respectively, at a detectable level if the eIF4G2 segment can bind to the 35S-labeled proteins at an affinity similar to those deduced in Fig. . Therefore, the results in Fig. and C even suggest that eIF1 and eIF5 cannot bind to eIF4G2 simultaneously. To test this idea directly, we conducted the binding assay between GST-eIF4G2439-914 and 35S-eIF5241-405 in the presence of different amounts of eIF1. As shown in Fig. , the presence of eIF1 in ~10-fold molar excess over GST-eIF4G2439-914 inhibited this interaction (lane 4). Thus, the interactions of the eIF4G2 HEAT domain with eIF1 and eIF5 appear to be mutually exclusive.
To test if eIF4G and eIF3c can bind to eIF1 simultaneously, we allowed GST-eIF3c1-156 to bind 35S-eIF4G2439-914 in the presence of recombinant eIF1. As shown in Fig. , eIF1 efficiently bound to GST-eIF3c1-156 (top panel) and formed a bridge between eIF3c and eIF4G2 (bottom panel). We also found that 35S-eIF4G2439-914 bound efficiently to GST-eIF1 in the presence of eIF3c1-156 in an amount 10-fold over that of GST-eIF1 (data not shown). Therefore, eIF4G and eIF3c can bind to eIF1 simultaneously. The results shown in Fig. suggest that eIF1 and eIF5 are tethered to eIF4G at different steps in the initiation pathway, while they are held together by the simultaneous interactions with eIF3c (see Discussion).
eIF4G1 and eIF4G2 immunoprecipitate with overproduced eIF1 and eIF5.
Next, we examined if eIF1 interacts with eIF4G in vivo. We previously reported that eIF1, eIF5, eIF2, and a small proportion of eIF4G1 immunoprecipitated with the HA epitope-tagged eIF3i (Tif34p) subunit (3
). However, the observed eIF4G interactions may be mediated by the 40S ribosomes to which HA-eIF3 likely binds. The small amount of eIF4G immunoprecipitated with HA-eIF3 suggests that the steady-state level of 48S complexes is very low compared to 43S complexes containing only the MFC components (3
). In order to increase the fraction of eIF1/eIF4G complexes free of the 40S ribosome, we overproduced eIF1 in a strain encoding HA-tagged eIF4G1 and tested if the overproduced eIF1 coimmunoprecipitated with anti-HA antibodies. As shown in Fig. , top panel, HA-eIF4G1 was specifically precipitated regardless of overexpression of eIF1 (top panel, lanes 2, 5, 8, and 11) together with the eIF4E subunit of eIF4F (Fig. , second panel). Importantly, eIF1, when overproduced, was specifically precipitated with HA-eIF4G1 (compare lanes 8 and 11). The amount of coimmunoprecipitated eIF1 corresponds to ~10% of the native level of eIF1 (compare lanes 7 and 11). As a negative control, immunoblotting with anti-eIF3g antibodies suggested that little or no eIF3 is associated with HA-eIF4G1 under these conditions (Fig. , bottom panel).
FIG. 4. Coimmunoprecipitation of HA-eIF4G1 with overproduced eIF1 and eIF5. WCEs prepared from transformants of KAY35 (encoding untagged eIF4G1 ) and YAS2136 (encoding HA-tagged eIF4G1 [Table ]) carrying YEplac195 (Vector), YEpU-SUI1 (eIF1), (more ...)
Using a yeast strain encoding HA-eIF4G2 as a sole source of eIF4G, we also found that a small but significant amount of eIF1 (~5% of the native level) precipitated with anti-HA antibodies, only when eIF1 was overexpressed (data not shown). Therefore, both eIF4G1 and eIF4G2 can specifically bind to eIF1 when the eIF1 concentration is raised in the cell. Because we did not detect the association of eIF1 with HA-eIF4G at the native level of eIF1, the observed interactions may not be physiological. However, we show below that overexpression of eIF1, and hence its increased interaction with eIF4G, has a physiological impact on the growth of some eIF4G mutants under certain conditions (see Fig. , below). Likewise, we found that eIF5 coprecipitated with HA-eIF4G1 when eIF5 was overproduced (Fig. ), suggesting that eIF5 binds to eIF4G not only in vitro (6
) but also in vivo.
FIG. 5. Genetic interaction between eIF4G2 and eIF1 or eIF5. Transformants of YAS1951 (WT), YAS1998 (tif4632-1), or YAS2002 (tif4632-430) carrying an appropriate URA3 plasmid were grown overnight in synthetic dextrose medium containing adenine (see Tables (more ...) Phenotypic competition between eIF4A and eIF1/eIF5 for an eIF4G2 Ts− mutant.
In order to examine the physiological relevance of the observed in vivo interactions between eIF4G and eIF1 or eIF5, we overproduced the latter in previously characterized temperature-sensitive (Ts−
) eIF4G2 mutant strains and examined the growth of the resulting strains at restrictive or semipermissive temperatures. These mutant strains contain chromosomal deletions of both eIF4G-encoding loci, TIF4631
, and harbor a single-copy plasmid encoding mutant eIF4G2 as the sole source of eIF4G. The tif4632-1
, and tif4632-8
mutations shown in Fig. confer Ts−
growth phenotypes that are suppressible by overproduced eIF4A (26
). We found that the overexpression of eIF1 or eIF5 did not affect the Ts−
growth of tif4632-1, tif4632-6
, and tif4632-8
mutants at either restrictive (37°C) or semipermissive (35°C) temperatures (Fig. , lines 4 and 5 for tif4632-1
; data not shown for tif4632-6
). However, overexpression of eIF4A restored the growth of these mutants (Fig. , line 3 for tif4632-1
; data not shown for tif4632-6
), as reported previously (26
Because the C-terminal half of eIF4G containing the HEAT domain interacts with eIF4A (21
) and also eIF1 and eIF5, we pondered whether overexpression of eIF1 or eIF5 would have a negative effect on the suppression of the tif4632
mutant phenotypes by overproduced eIF4A. To address this possibility, we constructed a plasmid overexpressing both eIF4A and eIF1 or eIF5, and we introduced it to the HEAT domain mutants mentioned above. We found that the suppression of tif4632-1
(Fig. , line 6), but not that of tif4632-6
(data not shown), by excess eIF4A was indeed reversed by cooverexpression of eIF1. We confirmed by immunoblot analyses that the level of eIF4A expression was not altered by cooverexpression of eIF1 (data not shown). To confirm that this effect is due to eIF1, we introduced a frameshift mutation, sui1Ω
, into the eIF1 open reading frame present in the cooverexpression plasmid (Table ). sui1Ω
is unconditionally lethal (data not shown). As expected, sui1Ω
in the cooverexpression plasmid restored the ability of intact eIF4A on the plasmid to suppress the tif4632-1
phenotype (Fig. , line 7). Likewise, the suppression of the tif4632-1
phenotype by eIF4A overexpression was reversed by cooverexpression of eIF5 (lines 8 and 9) without altering the level of eIF4A (data not shown). These results support the physiological relevance of eIF4G binding to overproduced eIF1 and eIF5 (Fig. ), providing firm genetic evidence that eIF4G2 can interact with eIF1 and eIF5 in vivo, presumably at the HEAT domain.
A second piece of genetic evidence for interaction between eIF4G2 and eIF1.
mutation altering Leu-428 and Leu-429 to alanines is located outside of the HEAT domain and is deficient in interaction between eIF4G2 and eIF4E (36
) (Fig. ). During the course of the study, we observed that overexpression of eIF1 exacerbated the weak Ts−
growth of the tif4632-430
mutant at the restrictive temperature (Fig. , line 14 versus 12). As eIF1 was overproduced from a high-copy plasmid carrying a 1.2-kb yeast chromosomal segment that contains other genes besides eIF1, we wished to verify that overexpression of eIF1 was responsible for this phenotype. For this purpose, we subcloned the eIF1 open reading frame under the GPD
promoter carried on a high-copy-number vector and used the resulting plasmid for overexpression of eIF1. As expected, the effect of this plasmid on growth of the tif4632-430
mutant was identical to that of the eIF1 plasmid used in the previous experiment (Fig. , line 16). We conclude that the overproduction of eIF1 reduces the growth of the eIF4G2 mutant, which is deficient for eIF4G2-eIF4E association. We also found that overexpression of eIF1 did not affect the weak Ts−
phenotypes of tif5-7A
impairing eIF5 (4
) or of tif34-1
impairing eIF3i (5
), indicating that the synthetic effect of overexpression of eIF1 is specific for tif4632-430
(data not shown).
We then wished to determine whether this negative effect of eIF1 can be eliminated by cooverexpression of eIF4A or eIF5. Cooverexpression of both eIF4A (line 17) and eIF5 (line 18) reversed the synthetic phenotype between tif4632-430 and excess eIF1. The effect of eIF5 overexpression on this synthetic phenotype (line 18) is consistent with our finding that the binding of eIF4G2 to eIF1 and eIF5 is mutually exclusive (Fig. to D); if association of the mutant eIF4G2 with eIF1 free of the ribosome is toxic, its prior association with eIF5 can prevent the toxic interaction due to the mutual exclusivity. Likewise, the counteractive effect of excess eIF4A on the synthetic phenotype (line 17) suggests that excess eIF4A outcompetes the binding of mutant eIF4G2 to eIF1 in vivo. Together with the effect of excess eIF1 on suppression of the tif4632-1 phenotype by excess eIF4A (Fig. , lines 6 and 7), the results suggest that the binding of eIF4A and at least eIF1 to the eIF4G HEAT domain is mutually exclusive. In conclusion, our results shown in Fig. provide a second piece of genetic evidence for in vivo interaction between eIF4G and eIF1.
The interactions of the eIF4G HEAT domain with eIF1 or eIF5 and eIF4A are not mutually competitive in vitro.
The genetic data shown in Fig. suggest that the interactions of eIF4G with eIF1 and eIF4A, and those of eIF4G with eIF5 and eIF4A, are mutually competitive. In order to examine whether eIF1 directly competes with eIF4A for binding to the eIF4G HEAT domain, we allowed GST-eIF1 to bind 35
in the presence of eIF4A in ~8-fold molar excess relative to GST-eIF1. Surface plasma resonance analyses indicate an equilibrium dissociation constant, Kd
, of 3.4 ± 0.2 μM for eIF4A binding to eIF4G2439-914
(data not shown). Thus, the binding of eIF1, eIF5, and eIF4A were of similar strengths in all in vitro assays performed. Accordingly, the eightfold excess of eIF4A over GST-eIF1 when added to the reaction should reduce the GST-eIF1-35
interaction ~10-fold at equilibrium if this interaction competes with the eIF4A-eIF4G2439-914
interaction. However, we found that this amount of eIF4A did not interfere with the interaction between GST-eIF1 and 35
(Fig. , lane 5 versus lane 3), suggesting that the two interactions are not competitive. As eIF4A is an ATP-binding protein, we also examined the effect of ATP in the competition assay described above. However, the presence of ATP (1 mM) in the reaction did not affect the outcome of the experiments (Fig. , lane 7). Similarly, we found that the binding of GST-eIF5241-405
to eIF4G does not compete with the eIF4G-eIF4A interaction in the presence or absence of ATP (Fig. , lanes 6 and 8). We also found that the binding of GST-eIF4G2439-914
(~1 μg) to 35
S-eIF1 was not inhibited by excess eIF4A (50 μg), even though eIF4A was present in large excess over GST-eIF4G2439-914
and can bind to the latter (data not shown). Therefore, the purified eIF4A did not compete with eIF1, or eIF5, for the interaction with the eIF4G2 HEAT domain under the conditions we examined. Because we confirmed that tif4632-1
completely abolishes GST-eIF4G2439-914
-eIF4A interaction in vitro (data not shown; see also reference 26
), we were not able to address the counteractive effect of eIF1 or eIF5 on this already weakened interaction. These results indicate that direct physical competition for binding to the HEAT domain may not underlie the functional competition between eIF1 or eIF5 and eIF4A observed in vivo. The idea of competition between eIF1 or eIF5 and eIF4A for eIF4G2 must be addressed in experiments including eIF4B, eIF3, or the entire eIF4G, either wild type or mutant (see Discussion).
FIG. 6. Purified eIF4A does not inhibit interaction between eIF4G2 HEAT domain and eIF1 or eIF5-CTD in vitro. 35S-eIF4G2439-914 was bound to GST fusion proteins (10 μg), indicated across the top, in 200 μl of binding buffer in the presence or (more ...) A known eIF4G2 HEAT domain mutation allows translation from a UUG codon.
So far, we showed that eIF4G interacts with eIF1 and eIF5 in vitro (Fig. to ) and in vivo (Fig. ), and we confirmed the biological significance of the in vivo interactions of eIF4G with overproduced eIF1 or eIF5 by using genetic approaches (Fig. ). Next we turned our attention to examining the direct role of the eIF4G HEAT domain in the 48S complex during stringent AUG selection. For this purpose, we tested if the known eIF4G2 mutations altering residues in the HEAT domain (Fig. ) allow increased translation from a non-AUG start codon. We transformed the tif4632-1
, and tif4632-8
mutants with a UUG-his4
plasmid and assayed β-galactosidase activity produced in each transformant. The UUG-his4
construct contains two UUG codons in frame with lacZ
, one at codon 1 and the other at codon 3 of his4
. It was shown that among non-AUG codons, UUG is used preferentially in different Sui−
). As shown in Fig. , the tif4632-1
(column 2) and tif4632-6
(column 3) mutants had 1.8- and 2.2-fold higher expression of UUG-his4
, respectively, than the isogenic wild-type strain. As a positive control, we found that a known Sui−
mutation in eIF5 (E. Hannig and C. Curtis, personal communications) led to 4.9-fold higher UUG-his4
expression than its isogenic wild type (Fig. , columns 5 and 6).
FIG. 7. Translation initiation from UUG codons in yeast eIF4G2 mutants. Transformants of YAS1951 (TIF4632+; WT; column 1), YAS1998 (tif4632-1; column 2), YAS1999 (tif4632-6; column 3), YAS2000 (tif4632-8; column 4), KAY113 (TIF5+; WT; column 5), (more ...)
In order to test whether the UUG-his4
expression observed with the tif4632
mutants depends upon the UUG start codon, we assayed his4
expression from a second reporter containing AUU and UUA at codons 1 and 3, respectively. As shown in Fig. , lacZ
expression from this reporter was ~60% of the values obtained with UUG-his4
in the wild-type strains (Fig. and B, columns 1 and 5) and was not altered at all by the tif4632
mutations or the control SUI5
mutation. (Note the same scale used for Fig. and B.) Therefore, the UUG codon appears to be preferentially used as a start codon in tif4632-1
6 mutants, as observed previously for a SUI5
To determine whether the increased UUG-his4 expression is accompanied by an increase in translation from the canonical AUG codon, the AUG-his4::lacZ plasmid containing AUG at codon 1 was also analyzed in parallel. As we used a synthetic complete (SC) medium to grow yeast prior to β-galactosidase assays, his4::lacZ transcription should not be derepressed by Gcn4p via the general amino acid control pathway. Consistently, we found that expression of AUG-his4::lacZ was relatively low and was not altered by tif4632-1 or tif4632-8 (Fig. , columns 1, 2, and 4). However, AUG-his4::lacZ translation was increased twofold with tif4632-6 (Fig. , column 3), suggesting that this mutation increases the frequency of initiation from both UUG and AUG codons. After normalizing for AUG-his4::lacZ expression, the UUG suppression activity (the percentage of the value in Fig. relative to that in C) still increased 1.9-fold in tif4632-1 compared to the isogenic wild type (Fig. ). (The mutant Sui− phenotype is more obvious in the experiment shown below in Table , as the UUG/AUG expression ratio for the vector control tif4632-1 transformant was 3.2-fold higher than that for the wild type.) The control SUI5 mutation increased the UUG suppression activity 5.2-fold (Fig. ). We also found that the tif4632-430 mutation, defective in eIF4E binding to eIF4G2, did not change LacZ expression from either the AUG-his4 or UUG-his4 reporter compared to wild type (data not shown). Thus, the tif4632-1 mutation specifically increases translation from a UUG codon and hence shows a mild Sui− phenotype. Since we grew tif4632 mutants at a permissive temperature prior to the assays, the observed phenotype should not be due to a secondary effect of limiting mRNA binding.
Effect of eIF1 and eIF5 overexpression on the mild Sui− phenotype of tif4632-1
We also tested if the mild Sui− phenotype observed with tif4632-1 is suppressible by overexpression of eIF1 and eIF5. Table summarizes the results of a β-galactosidase assay performed with tif4632-1 transformants carrying the his4::lacZ fusion plasmid and compatible eIF1 or eIF5 overexpression plasmid, together with appropriate control transformants. We found that overexpression of eIF1 or eIF5 did not alter the low UUG/AUG expression ratio (3.5 to 3.8%) of the wild-type strain (lines 1 to 6, column 6). Interestingly, overexpression of eIF1, but not that of eIF5, significantly reduced the UUG/AUG expression ratio that was increased threefold by tif4632-1 (lines 7 to 12, column 6). These results strongly suggest that a reduced interaction with eIF1, as observed in Fig. , at least partially accounts for the Sui− phenotype caused by tif4632-1.
A known Sui− mutation in eIF1 reduces its binding to eIF4G2 in vitro and in vivo.
Finally, we examined whether the reduced interaction between eIF4G and eIF1 could account for the phenotypes of previously known Sui−
mutants mapping in eIF1 (41
). For this purpose, we first conducted GST pull-down assays with an eIF1 mutant carrying sui1-1
(D83G). To our surprise, sui1-1
reduced the binding of 35
S-eIF1 to GST-eIF4G2439-914
by fourfold, but not its binding to GST-eIF3c1-156
(Fig. , top and second panel). As a control, GST-eIF4G2439-914
, but not GST-eIF3c1-156
, bound specifically to 35
S-eIF4A (third panel), confirming the previously identified interaction (26
). These results indicate that a known Sui−
mutation in eIF1 reduces its binding to eIF4G2 in vitro.
FIG. 8. The sui1-1 mutation in eIF1 reduces its binding to eIF4G2 in vitro and in vivo. (A) GST-fusion proteins listed across the top were assayed for binding to 35S-eIF1, -eIF1 carrying sui1-1, and -eIF4A, synthesized in rabbit reticulocyte lysates as described (more ...)
Next, we tested the effect of sui1-1 on the phenotype we observed with tif4632-430. We found that overexpression of the wild-type SUI1 allele, but not of the sui1-1 allele, exacerbated the weak Ts− phenotype of tif4632-430 (Fig. , lines 3 and 4). Immunoblotting with anti-eIF1 antibodies indicated that both SUI1 and sui1-1 alleles overproduce equivalent levels of eIF1 (Fig. ). Thus, the lack of synthetic phenotype with sui1-1 is due to its functional defect, presumably in the interaction with eIF4G. Based on these results, we conclude that the interaction of the eIF4G HEAT domain with at least eIF1 is important for the initiation complex to locate the first AUG codon accurately.